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Identification of the miRNA targetome in hippocampal neurons using RIP-seq

ABSTRACT: We established a neuron-specific Argonaute2:GFP-RNA immunoprecipitation followed by high throughput sequencing (AGO2-RIP-seq) to analyse the regulatory role of miRNAs in mouse hippocampal neurons. Using this technique, we identified more than two thousand miRNA target genes in hippocampal neurons, regulating essential neuronal features such as axon guidance and transcription. Furthermore, we found that stable inhibition of the highly expressed miR-124 in hippocampal neurons led to significant changes in the AGO2 binding of target mRNAs, resulting in subsequent upregulation of numerous miRNA target genes. Our data suggest that target redundancies are common among microRNA families. Together, these findings greatly enhance our understanding of the mechanisms and dynamics through which miRNAs regulate their target genes in neurons.

ORGANISM(S): Mus musculus

PROVIDER: GSE65543 | GEO | 2015/07/31

SECONDARY ACCESSION(S): PRJNA274334

REPOSITORIES: GEO

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Identification of the miRNA targetome in hippocampal neurons using RIP-seq

Project description:We established a neuron-specific Argonaute2:GFP-RNA immunoprecipitation followed by high throughput sequencing (AGO2-RIP-seq) to analyse the regulatory role of miRNAs in mouse hippocampal neurons. Using this technique, we identified more than two thousand miRNA target genes in hippocampal neurons, regulating essential neuronal features such as axon guidance and transcription. Furthermore, we found that stable inhibition of the highly expressed miR-124 in hippocampal neurons led to significant changes in the AGO2 binding of target mRNAs, resulting in subsequent upregulation of numerous miRNA target genes. Our data suggest that target redundancies are common among microRNA families. Together, these findings greatly enhance our understanding of the mechanisms and dynamics through which miRNAs regulate their target genes in neurons. Analysis of the miRNA targetome in hippocampal neurons after inhibition of 2 different miRNAs. AAV5 injections into the hippocampus of adult C57BL/6 mice producing either of the following under a synapsin promoter: GFP only (Samples beginning with 'GFP124…' or 'GFP125…'), GFP-miR124sp (Samples beginning with 'miR124…'), GFP-miR125sp (Samples beginning with 'miR125…'), GFP-AGO2-miR292sponge (samples ending with '…292'), GFP-AGO2-miR124sponge (samples ending with '…124'), GFP-AGO2-miR125sponge (samples ending with '…125'). All other samples were sham-injected.

2015-07-31 | E-GEOD-65543 | biostudies-arrayexpress

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Microarray analysis to identify miRNA targetome of primary Burkitt lymphoma tumors in comparison to normal CD19+ B cells

Project description:MicroRNAs (miRNAs) are small non-coding RNAs involved in the posttranscriptional regulation of gene expression. Deregulated miRNA levels have been reported in Burkitt lymphoma (BL) compared to normal B cells and a number of miRNA target genes have been identified in BL cell lines. Here, we determined for the first time the miRNA targetomes of primary BL tumors in comparison with normal B cells. AGO2-RNA immunoprecipitation (AGO2-RIP) in two frozen diagnostic BL tissue samples and three CD19+ B-cell samples isolated from routinely removed tonsils revealed distinct miRNA targetomes of BL and normal B cells. Targets of the miR-17~92 cluster were significantly more frequently AGO2-IP enriched in BL while targets of miR-29 and miR-150 were more frequently AGO2-IP enriched in normal B cells. Furthermore, we showed that miRNA target genes in BL are mainly involved in cell cycle and cell death. Immunohistochemistry on BL tumor samples and tonsil tissues confirmed altered protein levels for two out of six selected miRNA targets, in line with the differential AGO2-IP enrichment between BL and normal B cells. A comparison of AGO2-IP enriched genes in primary BL cases with BL cell lines indicated that approximately half of the miRNA targets may be missed in studies using BL cell lines, while almost 60% of miRNA target genes identified in cell lines were not AGO2-IP enriched in primary BL cases. Our results demonstrate both the necessity and feasibility of studying miRNA-target interactions in primary tumors.

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RNA-seq of miR-CLIP double purified, miR-124/miR-132 miR-CLIP probe treated HEK293T cells and corresponding mock treated and intermediate samples

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2020-11-23 | E-MTAB-8517 | biostudies-arrayexpress

Essential role for Argonaute2 protein in mouse oogenesis

Project description:Argonaute2 proteins (Ago2) are key component of RNA-induced gene silencing complexes (RISCs), which is crucial for microRNAs to repress target genes. The function of Ago2 for oogenesis is unknown due to early embryonic lethal of Ago2 knockout mice. Here we show the effect of loss of Ago2 in mouse oogenesis by specific deletion of it in growing oocyte. Although the Ago2-deficient oocytes can develop to mature oocytes, they have abnormal spindle and the chromosomes that can’t cluster together properly, which are very similar to the phenotype of Dicer-deficient oocytes. We checked miRNA expression profile and found that in Ago2-deficient oocyte, the expression of most miRNAs reduced more than 80%. To understand the downstream genes regulated by Ago2, we used microarray on Ago2-deficient oocyte and found that 512 genes were upregulated and 1073 genes were downregulated (FC>2, P<0.05). Our data proved that Ago2 have a key function for mouse oogenesis through globally regulating miRNA stability and function.

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Project description:MicroRNAs (miRNA) are small, non-coding RNAs mediating post-transcriptional regulation of gene expression. miRNAs have recently been implicated in hippocampus-dependent functions such as learning and memory, although the roles of individual miRNAs in these processes remain largely unknown. Here, we achieved stable inhibition using AAV-delivered miRNA sponges of individual, highly expressed and brain-enriched miRNAs; miR-124, miR-9 and miR-34, in hippocampal neurons. Molecular and cognitive studies revealed a role for miR-124 in learning and memory. Inhibition of miR-124 resulted in an enhanced spatial learning and working memory capacity, potentially through altered levels of genes linked to synaptic plasticity and neuronal transmission. In contrast, inhibition of miR-9 or miR-34 led to a decreased capacity of spatial learning and of reference memory, respectively. On a molecular level, miR-9 inhibition resulted in altered expression of genes related to cell adhesion, endocytosis and cell death, while miR-34 inhibition caused transcriptome changes linked to neuroactive ligand-receptor transduction and cell communication. In summary, this study establishes distinct roles for individual miRNAs in hippocampal function.

2016-03-01 | GSE68884 | GEO

A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity

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2010-07-01 | GSE21503 | GEO

Distinct cognitive effects and underlying transcriptome changes upon inhibition of individual miRNAs in hippocampal neurons

2016-03-01 | E-GEOD-68884 | biostudies-arrayexpress

Expression data from Dicer and Ago2-knockdown HeLa cells

Project description:Dicer and Argonaute2 (Ago2) gene is involving in microRNA (miRNA) maturation. Knockdown of these genes has great impact on miRNA expression profiles. We used microarrays to detail the miRNA expression profiles in Dicer- and Ago2-knockdown HeLa cells and demonstarted that the significant difference between Ago2-knockdown and Dicer- and Ago2-co-knockdown HeLa cells were not found.

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